Development of a Laboratory Based Method for Enumeration of Non-Culturable Indicator Fecal Coliform and E. coli in Thermophilic Anaerobically Digested Biosolids

Recent research has shown that a substantial, sudden increase in the indicator bacteria density, fecal coliform and E. coli, could occur immediately after centrifuge dewatering of thermally treated biosolids. One possible explanation is that the EPA promulgated MPN method used for enumeration (Method 1680) is underestimating the potentially viable bacteria in the sample prior to dewatering, and the dewatering process reactivates the bacteria such that they become culturable. The objectives of this research were to investigate alternative methods for enumerating indicator bacteria in thermally treated biosolids samples. The research approach used methods proposed in the literature for resuscitating bacteria that have become nonculturable after exposure to stress. For thermophilically treated biosolids samples that meet the EPA time-temperature requirements for Class A biosolids, the densities measured by the EPA 1680 Method were typically non-detect with detection limits less than 10 MPN/g DS. In comparison, the alternative or enhanced methods often had E. coli densities in the range of 1-4 log10 per gram dry solids. The results support the non-culturable hypothesis, however, additional work is needed to further verify the results and assure that the target organisms are being enumerated.